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An autoantibody to Rh-Ce protein causing positive direct antiglobulin test in a healthy blood donor

1 Department of Blood Bank, S. G. Shalby Hospital, Ahmedabad, Gujarat, India
2 Research Section, Lok Samarpan Raktadan Kendra and Research Centre, Surat, Gujarat, India

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Date of Submission13-Aug-2022
Date of Decision12-Sep-2022
Date of Acceptance18-Sep-2022
Date of Web Publication13-Dec-2022


A positive direct antiglobulin test (DAT) is of diagnostic feature for the patient with autoimmune hemolytic anemia (AIHA). However, on rare occasions, for obscure reasons, it is found among healthy blood donors. The present report is aimed to elucidate serological and immunological characteristics of such autoantibody in a healthy donor aged 62 years found with positive DAT. There was no history of Leishmaniasis, nor having a significant illness. His red blood cells (RBCs) showed incompatible cross-match results with every recipient tested in the antiglobulin phase. He was found to be DAT+. As his plasma had very little presence of autoantibody, hence was augmented by elution from his in vivo sensitized RBCs for the study. Autoantibody with immunoglobulin IgG showed predominant specificity of anti-Ce. It is certainly a rare case of autoantibody to RhCe compound antigen yet being innocuous in a healthy blood donor with a positive DAT.

Keywords: Autoanti-Ce antibody, healthy blood donor, positive direct antiglobulin test

How to cite this URL:
Parikh YN, Vekariya MM, Gondaliya VK, Rajapara MM, Joshi SR. An autoantibody to Rh-Ce protein causing positive direct antiglobulin test in a healthy blood donor. Asian J Transfus Sci [Epub ahead of print] [cited 2023 Jan 28]. Available from:

   Introduction Top

A positive direct antiglobulin test (DAT) has diagnostic significance for autoimmune hemolytic anemia (AIHA). However, on rare occasions, it is found among healthy blood donors with no sign of clinical disease. While occurring in a frequency of 1:13,000 among healthy individuals,[1] its cause remains obscure with speculations as to being associated with certain infections such as malaria[2] or kala-azar[3],[4] or due to premalignant hematological conditions.[5] A presence of complement fraction on the RBC membrane may indicate the complement-fixing immunoglobulin M cold agglutinins or if the immunoglobulin G (IgG) molecule is detected it suggests the warm temperature reacting autoantibodies. In the latter case, serological specificities are found against the antigens of the Rh blood groups.[6] Sometimes autoantibodies with a high affinity to the antigen are found only on the red blood cells (RBCs) membrane with little presence of free antibodies in circulating plasma. In such a situation, one needs to elute the IgG autoantibody molecules from the RBCs for determining the serological specificity. The aim of the present study was to understand the serological and immunoglobulin nature of the autoantibody found in a healthy donor showing a strong DAT+.

   Materials and Methods Top

A donor's blood samples and the eluate prepared from his red cells were used to carry out various serological tests. Rh subtyping was carried out using commercial antisera (Tulip Diagnostics, India) reacting in the saline tube phase. Reagents such as papain and phosphate-buffered saline (PBS) were prepared in house. Standard protocol was followed for antibody identification using a commercial cell panel (Immucor, USA). Ether elution method was used to augment antibodies from the donor's RBCs already sensitized in vivo. A slight modification in testing the eluate was employed to avoid interference in reading the results due to the dense hemoglobin (Hb) present in the eluate prepared as described earlier.[7] In brief, the reagent RBCs, the eluate, and the low-ionic-strength solution were mixed and incubated in a tube at 37°C for 15 min. The sensitized RBCs washed using PBS were superimposed onto the gel card and centrifuged, and read the result.

   Results Top

The case

A 62-year-old male healthy blood donor never transfused nor had any significant medical history except surgery for a kidney stone. There was no history of Malaria or kala-azar, although he hailed from the region known for kala-azar prevalence. He used to take alprazolam whenever required. Three months before blood donation, he had received the prophylactic dose of the COVID-19 vaccine. He passed through all the criteria for blood donation with parameters such as Hb, 13.0 g/dl; blood pressure, 142/80 mmHg; pulse, 90/min; body temperature, 98.2°F. His peripheral reticulocyte count was found to be 2%. His health status remained uneventful throughout 1 year of the follow-up.

Grouped as B, RhD+ (Rh-subtype Dce/Dce, R1R1), his RBCs were found incompatible in the antiglobulin phase during crossmatch test with several homologous recipients. The DAT on his RBCs was positive with the broad-spectrum antiglobulin reagent explaining the incompatibility with the serum/plasma of every recipient tested. His serum/plasma showed very little antibody and the DAT was positive by monospecific anti-IgG but not by anti-C3d, the antibody was eluted from his sensitized RBCs for study purposes. The antibody tested with the RBC panel showed anti-E-like specificity as it reacted with the cells having E antigen but not with those lacking the antigen E [Figure 1]. Interestingly, the eluate tested by the conventional tube method reacted stronger (Grade 4+) with the RBC typed R1R1 (DCe/DCe) as compared to that typed rr (dce/dce) in Grade 3–2+ that lead us to perform a titration of the antibody using selected RBCs. The RBCs typed R1R1 (DCe/DCe) showed a titer value of 1:64, whereas those with rr (dcd/dce) type showed the titer of 1:4, thereby indicating the preferential specificity as anti-Ce (anti-Rh1). However, reactivity against the RBC typed rr (dcd/dce) was not separable from that against the RBCs typed R1R1 (DCe/DCe) by the selective adsorption method used.
Figure 1: Results on the eluate tested with red cell panel

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   Discussion Top

A positive DAT on RBCs with broad-spectrum antiglobulin reagent is associated with acquired hemolytic anemia with free autoantibody present in circulating plasma.[8] The use of monospecific antiglobulin reagents is of diagnostic significance as the cold agglutinin disease usually shows a presence of complement fraction on to the RBCs, whereas the other autoimmune conditions involved with the warm temperature reacting autoantibody may show the presence of IgG molecules on the cell surface, thus providing a differential diagnosis and thereby a line of treatment to be administered to the patients. Some rare healthy individuals with a positive DAT were found in a frequency of around 1:13,000,[1] although a higher incidence of 1:1000 donors was also shown when the cases showing weakly positive DAT were included.[9] Antibody in the majority of such donors, including that in the present study, showed the immunoglobulin subclass as IgG1 which is otherwise known to cause clinical hemolysis, yet remained innocuous for unknown reasons. Many times, such healthy donors with positive DAT are noticed during crossmatch tests in the antiglobulin phase[1],[10] as was the case with our donor.

Serological specificity of the autoantibody involved was often related to anti-Rh along with anti-Wrb.[6] Autoantibody specificity in the present case was also found against the RH-Ce protein. While gel column technology showed an anti-E specificity of the autoantibody and the manual tube test, followed by titration of antibody clearly showed differential strength of agglutination with the RBCs typed R1R1 (DCe/DCe) and rr (dce/dce), thereby indicating to the dual specificities as anti-Ce (-Rh1) and anti-E, although both the entities were not separable by selective absorption. This observation reflects on the limitation of the advanced gel card technology against the age-old tube method in distinguishing different specificities in antibody identification exercises. In one study, involving a large number of cases, numbering 150, with positive DAT among the patients with AIHA and α-methyldopa in-takers along as well as the healthy individuals, the authors had observed only one case with the specificity of anti-Ce alongside other specificities that too in the patients having AIHA.[6] Anti-Ce as autoantibody found in normal healthy donors, therefore, merits as the first case of its kind ever to be documented in the literature.

There are scanty reports in the literature on normal individuals with a positive DAT due to the presence of complement or IgG on the RBCs.[1],[6],[9] A positive DAT associated with malaria was presumed to be a process in the development of immunity to malaria[2] or as nonspecific adsorption of IgG/complement on RBCs in conditions like kala-azar.[3],[4] However, the present case did not give a recent history of malaria, although a possibility of the asymptomatic subclinical infection of leishmaniasis still remains open as he hails from the region where the endemic kala-azar has been documented.[11] While a positive DAT without any evidence of a hemolytic process is found in patients taking α-methyldopa or a variety of other drugs,[6] the present case had no history of taking α-methyldopa, although he used to take anti-depressant medication whenever required, this drug is not known to cause a positive DAT. The DAT+ healthy individuals have a risk of developing AIHA[1] or hematologic malignancy.[5] However, our donor remained in excellent health even after 1 year of the follow-up period.

   Conclusion Top

A rare case of a healthy blood donor with positive DAT by IgG1 anti-Ce is described. While the case of anti-Ce autoantibody associated with AIHA has been documented, anti-Ce as an autoantibody in healthy individual merits the first of its kind in the available literature.

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Conflicts of interest

There are no conflicts of interest.

   References Top

Gorst DW, Rawlinson VI, Merry AH, Stratton F. Positive direct antiglobulin test in normal individuals. Vox Sang 1980;38:99-105.  Back to cited text no. 1
Abdalla S, Weatherall DJ. The direct antiglobulin test in P. Falciparum malaria. Br J Haematol 1982;51:415-25.  Back to cited text no. 2
Vilela RB, Bordin JO, Chiba AK, Castelo A, Barbosa MC. RBC-associated IgG in patients with visceral leishmaniasis (kala-azar): A prospective analysis. Transfusion 2002;42:1442-7.  Back to cited text no. 3
Woodruff AW, Topley E, Knight R, Downie CG. The anaemia of Kala-Azar. Br J Haematol 1972;22:319-29.  Back to cited text no. 4
Rottenberg Y, Yahalom V, Shinar E, Barchana M, Adler B, Paltiel O. Blood donors with positive direct antiglobulin tests are at increased risk for cancer. Transfusion 2009;49:838-42.  Back to cited text no. 5
Issitt PD, Pavone BG, Goldfinger D, Zwikder H, Issitt CH, Tessel JA, et al. Anti-Wrb, and other autoantibodies responsible for positive direct antiglobulin tests in 150 individuals. Br J Haematol 1976;34:5-18.  Back to cited text no. 6
Joshi SR, Vekariya M, Rajapara M. A modified approach in testing the ether eluate to overcome the hurdle in reading the results. Asian J Transfus Sci 2022. [In Press]  Back to cited text no. 7
Klein HG, Anstee DJ. Mollison's Blood Transfusion in Clinical Medicine. 12th ed. Oxford, UK: Wiley Blackwell; 2014.  Back to cited text no. 8
Allan J, Garratty G. Positive Direct Antiglobulin Tests in Normal Blood Donors (Abstract). 16th ed. Montreal: Congress of the International Society of Blood Transfusion; 1980.  Back to cited text no. 9
Puri V, Chhikara A, Sharma G, Sehgal S, Sharma S. Critical evaluation of donor direct antiglobulin test positivity: Implications in cross-matching and lessons learnt. Asian J Transfus Sci 2019;13:70-2.  Back to cited text no. 10
[PUBMED]  [Full text]  
Saha P, Ganguly S, Chatterjee M, Das SB, Kundu PK, Guha SK, et al. Asymptomatic leishmaniasis in kala-azar endemic areas of Malda district, West Bengal, India. PLoS 2017; Negl Trop Dis 11 (2): e0005391.  Back to cited text no. 11

Correspondence Address:
Sanmukh Ratilal Joshi,
Lok Samarpan Raktadan Kendra and Research Centre, Mini Bazar, Varachha Road, Surat - 395 006, Gujarat
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ajts.ajts_106_22


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